Microorganisms are used for industrial production of various useful substances, which include foods like alcoholic beverages, miso, and soy sauce, and also amino acids, organic acids, nucleic acid-related substances, antibiotics, saccharides, lipids, and proteins. These substances have wide ranging applications, such as in foods, drugs, detergents, cosmetics, and other items of daily use, and as raw materials for various chemical products.
Among microorganisms, gram-positive bacteria like Bacillus subtilis, Bacillus lichenifonnis, and Bacillus megaterium have a very high capacity to secrete a variety of exoenzymes such as amylases, proteases, and lipases. In fact, many exoenzymes produced by bacilli are presently in industrial use. Production of certain proteins by bacteria as secretions is useful in, for instance, that the secreted protein normally has its native structure, and the ease with which the secreted protein can be purified. Therefore, it is very meaningful to improve such bacterial strains in order to increase the amount of a given secretory protein secreted and produced.
Many of the proteins localized in the outer membrane and the periplasmic space of prokaryotes have passed through the cytoplasmic membrane mediated by a Sec translocation machinery. A transmembrane channel made of a heterotrimeric membrane protein complex consisting of SecY/SecE/SecG and a peripherally bound dimeric SecA, weakly bound to a SecD/SecF/YajD heterotrimeric membrane protein complex, is created, and the protein is translocated through the membrane with the help of energy from ATP hydrolysis by SecA. It is known that if the bacterium is Escherichia coli, the presecretory protein is recognized by the molecular chaperone SecB, and passed on to the SecA on the surface of the cytoplasmic membrane. The factor homologous with the molecular chaperone SecB has not yet been identified in Bacillus subtilis but it is believed that Bacillus subtilis has an SRP (signal recognition particle), characteristically involved in translocation across the endoplasmic reticulum membrane of eukaryotes, and that the secretory protein is handed over from the SRP to SecA. It has been shown that SecA of Bacillus subtilis consists of two domains, the N domain and the C domain, that the N domain has an ATP binding site I (ABS I) and a signal peptide binding site, that the C domain has sites that take part in SecA dimerization and interaction with SecY, and a region homologous with the SecB binding site of Escherichia coli, and that an ATP binding site II (ABS II) is present in a manner that aligns the two domains.
So far, genetic modifications such as deletion of a protease (Non-patent Document 1), enhancement of PrsA production (Non-patent Document 2), overexpression of SecD/SecE/SecDF (Patent Document 1), and overexpression of SecG (Patent Document 2) have been reported as techniques for increasing secretory production of proteins.
However, there has been no report on deletion of some of the amino acid residues of SecA for increasing secretory production of proteins.
[Non-patent Document 1] Olmos-Soto J, Contreras-Flores R. Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis. Appl. Microbiol. Biotechnol. 2003 September; 62(4):369-73.
[Non-patent Document 2] Vitikainen M, Hyyrylainen H L, Kivimaki A, Kontinen V P, Sarvas M. Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation. J Appl Microbiol. 2005; 99(2):363-75.
[Patent Document 1] WO99/04007
[Patent Document 2] WO99/04006